mmg_233_2013_genetics_genomicswikiaorg-20200214-history
Horizontal Gene Transfer in Clostridium difficile
Introduction The major nosocomial pathogen, Clostridium difficile, is known as the agent responsible for antibiotic-associated diarrhea (CDAD) via antibiotic disruption of the normal gut microflora and subsequent C. difficile establishment (Bartlett, 2008). The diarrhea produced by this pathogen results from the production of two toxins, A and B, that act as virulence factors disrupting the gut epithelium (Kuehne et al., 2010; Lyras et al., 2009) and can range from mild to potentially fatal due to complications such as toxic megacolon (Rupnik, Wilcox, & Gerding, 2009). These two toxins are encoded for within the pathogenicity locus (PaLoc) region that also contains regulatory genes and is not found in non-toxic pathogen strains. Conjugative transposons (CTns) are genetic elements that are usually inserted into a genome but maintain the capability to mediate their own transfer to complimenting recipients (Roberts & Mullany, 2011). C. difficile 630Δerm is a derivative of the toxic C. difficile 630 (CD630) strain that is sensitive to erythromycin, contains the PaLoc region (Kuehne et al., 2010; Lyras et al., 2009), and contains at least six active CTns. Conjugative transfer was previously observed for CTn1, CTn2, CTn4, CTn5, CTn7 and Tn5397, all of which have the potential for transformation to the non-toxic C. difficile 37 (CD37) strain (M. S. M. Brouwer, Warburton, Roberts, Mullany, & Allan, 2011; Mullany et al., 1990; Wang et al., 2000). This investigation aimed to determine the presence (or lack thereof) of any additional genetic information cotransferred to CD37 with the CTns (M. Brouwer et al., 2013). Methods To begin, nine CD37 transconjugants containing CTn1::ermB (an erythromycin-resistant derivative of CTn1 (M. S. M. Brouwer et al., 2011)) were investigated to determine the presence (or lack thereof) of additional cotransferred genetic elements. This investigation was performed via DNA isolation, amplification, and subsequent sequencing. To further determine whether newly acquired toxin genes were capable of mediated toxin production, a PaLoc-containing transconjugate that had acquired the wild-type PaLoc (PaLoc386) was subjected to a cytotoxicity assay with HFF-1 cells to determine potential cytopathic effects (CPE). Subsequent addition of a commercial TcdB antiserum showed abolishment of the CPE within both the 630Δerm and PaLoc386, indicating production of a functional toxin B from both strains. The underlying PaLoc transfer was then investigated via the determination of whole-genome sequences for seven transconjugants containing the marked or wild-type PaLoc. To further define the region that was horizontally transferred in the transconjugants, distinctions had to be made regarding the donor and recipient genome. In doing this, 30 regions of approximately 10 kb spaced evenly throughout the 630Δerm genome were selected and compared to the corresponding regions within the CD37 genome (M. S. M. Brouwer, Allan, Mullany, & Roberts, 2012). By aligning the genomic DNA sequence of the PaLoc-containing transconjugants in all 30 differing regions, it was observed that SNPs and indels allowed for the desired discrimination. The distance from the first SNP/indel upstream of the PaLoc to the last SNP/indel downstream of the PaLoc (specific to the donor strain (630Δerm)) was calculated to allow for an approximation of the length of transferred DNA in each transconjugate. This analysis could only provide a maximum and minimum size of the transferred fragment, however, as the high level of sequence identity between the two strains prevented further analysis. The same mating experiments were then repeated in the presence of DNase to exclude the possibility of transformation as a mechanism of transfer. Strain CD37 was then infected with phage suspensions made by mitomycin C induction of 630Δerm, however no plaques were observed. To confirm the presence of the infectious phages, another CD strain was utilized, CD843. Another study (Dingle et al., 2011) analyzed the molecular epidemiology of C. difficile strains and has suggested that PaLoc transfer is currently occurring in wild populations. To confirm this, two of the non-toxic isolates were tested to determine if they were able to act as recipients for the observed PaLoc in matings using 630Δerm as a donor. In doing this, spontaneous rifampicin-resistant derivatives of these strains were first established and the PaLoc was subsequently transferred. Results/Discussion This horizontal gene transfer study provided data to demonstrate the capability of the PaLoc in C. difficile 630Δerm in transferring to non-toxic strains via a conjugation-like mechanism. It was shown that in the case of CD37, transfer resulted in conversion to a toxin producer, indicating important clinical aspects for future research as non-toxic strains have shown success in treating CDAD (Villano, Seiberling, Tatarowicz, Monnot-Chase, & Gerding, 2012). Also of considerable importance is the finding that PaLoc transfer almost always occurs with the cotransfer of conjuative transposons that encode antibiotic resistance. This cotransfer of transposons results in the corresponding cotransfer of virulence and antibiotic-resistant genes (illustrated by the PaLoc transfer in PaLoc 386 in which Tn5397 encodes tetracycline resistance). Although transfer of the PaLoc and CTns occur at a low frequency, cotransfer of unselected genetic material with the PaLoc was observed. This can most likely be explained by the fact that one or more CTns initiates a transfer and at the same time other cellular elements (in the same cell) are activated by trans-acting regulatory molecules (such as that observed between Tn91- elements (Flannagan & Clewell, 1991)). Since the incoming chromosomal DNA that is integrated into a chromosomal backbone via homologous recombination is mediated by high-frequency recombination (Hfr), the entire chromosome could theoretically be transferred (i.e. all the genetic material of the chromosome along with the PaLoc). This rarely happens, however, due to ‘knicking’ of the incoming DNA strand that promotes a higher frequency of transfer for certain segments rather than the whole. Entire-genome sequencing has shown that large sections of DNA have transferred between C. difficile strains, thus indicating the importance of this form of transfer in evolution of C. difficile (He et al., 2010). References Bartlett, J. G. (2008). 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